RESUMEN
The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.
Asunto(s)
Hidroxilisina , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa , Colágeno/química , Colágeno/metabolismo , Compuestos Ferrosos , Lisina/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/antagonistas & inhibidores , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/químicaRESUMEN
Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid ß peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to â¼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.
Asunto(s)
Endosomas/metabolismo , Fosfatos de Inositol/metabolismo , Insulisina/metabolismo , Fosfatidilinositoles/metabolismo , Androstadienos/farmacología , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Endosomas/efectos de los fármacos , Activación Enzimática , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Insulisina/química , Insulisina/genética , Liposomas/química , Liposomas/metabolismo , Mutación , WortmaninaRESUMEN
Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli- or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
Asunto(s)
Ácidos Cetoglutáricos/química , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/biosíntesis , Animales , Células CHO , Carbono/química , Dióxido de Carbono/química , Cromatografía Liquida , Colágeno/química , Cricetulus , Medios de Cultivo Condicionados/química , Glicosilación , Humanos , Luciferasas/química , Lisina/química , Proteínas Recombinantes/biosíntesis , Ácido Succínico/química , Espectrometría de Masas en TándemRESUMEN
Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.
Asunto(s)
Proteínas de Arabidopsis/química , Fosfatasas de Especificidad Dual/química , Glucanos/química , Glucosa/química , Almidón/química , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Carbohidratos/química , Dominio Catalítico , Clonación Molecular , Fosfatasas de Especificidad Dual/metabolismo , Fosfatos/química , Fosforilación , Hojas de la Planta/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.
Asunto(s)
Membrana Celular/metabolismo , Neuropilinas/metabolismo , Secuencias de Aminoácidos , Animales , Axones/metabolismo , Adhesión Celular , Humanos , Ligandos , Ratones , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Semaforinas/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase like sex four2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Fosfatasas de Especificidad Dual/metabolismo , Glucanos/metabolismo , Almidón/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Cristalografía por Rayos X , Fosfatasas de Especificidad Dual/química , Fosfatasas de Especificidad Dual/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oligosacáridos/química , Oligosacáridos/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.
Asunto(s)
Calcineurina/metabolismo , Calmodulina/metabolismo , Secuencia de Bases , Calcineurina/química , Calmodulina/química , Cromatografía en Gel , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Humanos , Conformación Molecular , Espectrometría de FluorescenciaRESUMEN
Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Endotelio Vascular/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/metabolismo , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/genética , Animales , Unión Competitiva , Células Cultivadas , Furina/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuropilina-1/biosíntesis , Neuropilina-1/genética , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Sus scrofa , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/agonistas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Glucanos/metabolismo , Almidón/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Cristalografía por Rayos X , Fosfatasas de Especificidad Dual/química , Fosfatasas de Especificidad Dual/genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Lysyl hydroxylase 2 (LH2) catalyzes the formation of highly stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), thus promoting lung cancer metastasis through its capacity to modulate specific types of collagen cross-links within the tumor stroma. Using 1 and 2 from our previous high-throughput screening (HTS) as lead probes, we prepared a series of 1,3-diketone analogues, 1-18, and identified 12 and 13 that inhibit LH2 with IC50's of approximately 300 and 500 nM, respectively. Compounds 12 and 13 demonstrate selectivity for LH2 over LH1 and LH3. Quantum mechanics/molecular mechanics (QM/MM) modeling indicates that the selectivity of 12 and 13 may stem from noncovalent interactions like hydrogen bonding between the morpholine/piperazine rings with the LH2-specific Arg661. Treatment of 344SQ WT cells with 13 resulted in a dose-dependent reduction in their migration potential, whereas the compound did not impede the migration of the same cell line with an LH2 knockout (LH2KO).
RESUMEN
The neuropilin (Nrp) family consists of essential multifunctional vertebrate cell surface receptors. Nrps were initially characterized as receptors for class III Semaphorin (Sema3) family members, functioning in axon guidance. Nrps have also been shown to be critical for vascular endothelial growth factor-dependent angiogenesis. Intriguingly, recent data show that Nrp function in these seemingly divergent pathways is critically determined by ligand-mediated cross-talk, which underlies Nrp function in both physiological and pathological processes. In addition to functioning in these two pathways, Nrps have been shown to specifically function in a number of other fundamental signaling pathways as well. Multiple general mechanisms have been found to directly contribute to the pleiotropic function of Nrp. Here we review critical general features of Nrps that function as essential receptors integrating multiple molecular cues into diverse cellular signaling.
Asunto(s)
Neuropilinas/fisiología , Animales , Humanos , Integrinas/metabolismo , Ligandos , Neoplasias/irrigación sanguínea , Neoplasias/fisiopatología , Neovascularización Patológica , Neovascularización Fisiológica/efectos de los fármacos , Neuropilinas/química , Receptores de Superficie Celular/fisiología , Semaforinas/metabolismo , Transducción de Señal/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.
Asunto(s)
Colágeno Tipo I , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Hidroxilación , Lisina/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)-myosin IIA-containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a-dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8+ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Aparato de Golgi/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteínas de Transporte Vesicular/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Línea Celular Tumoral , Aparato de Golgi/genética , Aparato de Golgi/patología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Metástasis de la Neoplasia , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change.
Asunto(s)
Endosomas/metabolismo , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Pulmonares/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Actinas/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora , Adenocarcinoma del Pulmón/metabolismo , Línea Celular Tumoral , Polaridad Celular , Citoesqueleto/metabolismo , Endocitosis , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Cinesinas , Neoplasias Pulmonares/genética , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Metástasis de la NeoplasiaRESUMEN
Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.
Asunto(s)
Adenocarcinoma del Pulmón/fisiopatología , Progresión de la Enfermedad , Glucosiltransferasas/metabolismo , Neoplasias Pulmonares/fisiopatología , Animales , RatonesRESUMEN
Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Fibroblastos Asociados al Cáncer/metabolismo , Células Epiteliales/metabolismo , Neoplasias Renales/genética , Neoplasias Pulmonares/genética , Células Madre Mesenquimatosas/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/secundario , alfa-Globulinas/genética , alfa-Globulinas/metabolismo , Animales , Fibroblastos Asociados al Cáncer/patología , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Receptor con Dominio Discoidina 2/genética , Receptor con Dominio Discoidina 2/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/secundario , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Ratones Transgénicos , Transducción de Señal , Microambiente Tumoral/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Collagenous stromal accumulations predict a worse clinical outcome in a variety of malignancies. Better tools are needed to elucidate the way in which collagen influences cancer cells. Here, we report a method to generate collagenous matrices that are deficient in key post-translational modifications and evaluate cancer cell behaviors on those matrices. We utilized genetic and biochemical approaches to inhibit lysine hydroxylation and glucosylation on collagen produced by MC-3T3-E1 murine osteoblasts (MC cells). Seeded onto MC cell-derived matrix surface, multicellular aggregates containing lung adenocarcinoma cells alone or in combination with cancer-associated fibroblasts dissociated with temporal and spatial patterns that were influenced by collagen modifications. These findings demonstrate the feasibility of generating defined collagen matrices that are suitable for cell culture studies.
RESUMEN
Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Cromosomas Humanos Par 1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenocarcinoma del Pulmón/genética , Animales , Cromosomas Humanos Par 1/genética , Ensayo de Inmunoadsorción Enzimática , Aparato de Golgi/metabolismo , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Microtomografía por Rayos XRESUMEN
Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/antagonistas & inhibidores , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded ß-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer.